A suitable and efficient optimization system for the culture of Chlamydia trachomatis in adult inclusion conjunctivitis

Abstract The prevalence of Chlamydia trachomatis infection in the genitourinary tract is increasing, with an annual rise of 9 million cases. Individuals afflicted with these infections are at a heightened risk of developing adult inclusive conjunctivitis (AIC), which is commonly recognized as the ocular manifestation of this sexually transmitted infection. Despite its significant clinical implications, the lack of distinctive symptoms and the overlap with other ocular conditions often lead to underdiagnosis or misdiagnosis of AIC associated with C. trachomatis infection. Here, we established six distinct C. trachomatis culture cell lines, specifically highlighting the MA104 N*V cell line that exhibited diminished expression of interferon regulatory factor 3 (IRF3) and signal transducer and activator of transcription 1 (STAT1), resulting in reduced interferons. Infected MA104 N*V cells displayed the highest count of intracytoplasmic inclusions detected through immunofluorescence staining, peaking at 48 h postinfection. Subsequently, MA104 N*V cells were employed for clinical screening in adult patients diagnosed with AIC. Among the evaluated cohort of 20 patients, quantitative PCR (qPCR) testing revealed positive results in seven individuals, indicating the presence of C. trachomatis infection. Furthermore, the MA104 N*V cell cultures derived from these infected patients demonstrated successful cultivation and replication of the pathogen, confirming its viability and infectivity. Molecular genotyping identified four distinct urogenital serovars, with serovar D being the most prevalent (4/7), followed by E (1/7), F (1/7), and Ia (1/7). This novel cellular model contributes to studies on C. trachomatis pathogenesis, molecular mechanisms, and host–pathogen interactions both in vitro and in vivo. It also aids in acquiring clinically relevant strains critical for advancing diagnostics, treatments, and vaccines against C. trachomatis.


Introduction
Tr ac homa, an ancient infectious k eratoconjuncti vitis, was the leading pr e v entable infectious blinding eye disease globally, especiall y in de v eloping countries (Phillips 2019 ).In 2002, the World Health Organization reported that there were about 84 million people suffering from active trachoma worldwide (Mariotti et al. 2009, Taylor et al. 2014 ).This condition is caused by Chlamydia trachomatis infection, a significant pathogen also implicated in sexuall y tr ansmitted diseases (Pekmezovic et al. 2019 ).It is noteworthy that, despite a notable decline in tr ac homa incidence due to impr ov ed sanitation in recent decades, there has been a surge of 9 million new cases per year in urogenital C. trachomatis infections.Individuals with this infection face an ele v ated risk of adult inclusion conjunctivitis (AIC) (Darville andHiltke 2010 , Mohamed-Noriega et al. 2015 ).AIC is gener all y consider ed to be the ocular manifestation of this sexually transmitted infection (Postema et al. 1996 ).Clinicall y, AIC typicall y pr esents as c hr onic follicular conjunctivitis, c har acterized by an acute or subacute onset that may persist for months if left untr eated (Stenber g and Mårdh 1990 ).The absence of pathognomonic symptoms often results in the underdiagnosis or misinterpretation of AIC associated with C. trachomatis infection.
Labor atory tests ar e essential to determine the causative agent and confirm the clinical diagnosis, including Giemsa staining, dir ect fluor escent antibody (DFA) test, pol ymer ase c hain r eaction (PCR), and cell culture (Cook et al. 2005 ).The nucleic acid test is widely utilized for detecting urogenital C. trachomatis infection and chlamydial pneumonia due to its high diagnostic sensitivity (Solomon et al. 2004, Ozüberk et al. 2013 ).Ne v ertheless, labor atory diagnosis of AIC is challenging due to the limited specimen, often leading to minimal amounts of nucleic acids within clinical samples .Con v entional methods suc h as PCR, cell culture, and DFA testing have exhibited remarkably low rates of positive results.In a specific study, only 4 (8%) out of 50 conjunctival swab specimens tested positive using DFA, while only 1 (2%) sample yielded positive results through cell culture and PCR techniques (Solomon et al. 2004 ).Ther efor e, in vitro cultur e of C. trac homatis r epr esents a promising approach to improve the diagnostic sensitivity for AIC, serving as the gold standard for C. trachomatis diagnosis and providing the foundation for in vitro drug susceptibility test and further investigations of C. trachomatis pathogenesis (Di Pietr o et al. 2019 ).Mor eov er, the de v elopment of v accines r elies on liv e C. trac homatis str ains.Pr oper location and corr ect oper ation of biopsy, and exploring an optimal C. trachomatis culture system are fundamental to C. trachomatis culture and achieving accurate diagnosis of AIC.
Chlam ydia trac homatis is a strictl y intr acellular par asite that can onl y gr ow and r eplicate within host cells and cannot be cultured in vitro (Chiarelli et al. 2020 ).It can be classified into 20 genotypes based on the outer membrane gene (ompA), which is typically associated with distinct clinical presentations.Genotypes A-C (variant Ba) cause trachoma, while genotypes D-K (variant Da, Ga, and Ia) are linked to urogenital infections, and L1-L3 (variant L2b and L2c) genotypes cause l ymphogr anuloma v ener eum.Successful isolation of C. trachomatis relies on permissive cell lines, primarily epithelial cells like McCoy, HeLa, African green monkey kidney cells (Vero), and Buffalo green monk e y kidne y cells (BGMK).MA104 cells, an epithelial cell derived from African green monk e y kidney and known for their efficient culture of entero viruses , ha ve diminished capacity of producing interferon (IFN) upon infection (Lee et al. 2013, Feng et al. 2019, Nurdin et al. 2023 ).Ther efor e, MA104 cells may be used to cultur e C. trac homatis because it can elicit an innate immune response similar to that of viruses.
IFN signaling pathway plays a pivotal role in inhibiting the pr olifer ation and spr ead of C. trac homatis , acting indir ectl y by depleting intracellular tryptophan r eserv es and stim ulating indoleamine-2,3-dio xygenase acti vity to restrict the tryptophan suppl y of C. trac homatis (Islam et al. 2018, Wang et al. 2022 ).A notable disparity between urogenital and ocular C. trachomatis strains lies in the presence of tryptophan synthetase, enabling ur ogenital str ains to facilitate tryptophan synthesis through indole and sustain their normal life c ycle (Caldw ell et al. 2003, Carlson et al. 2004 ).Despite MA104 cells having a blunted IFN response, making them highly permissive for chlamydial replication, we hypothesize that deregulation of IFN signaling in MA104 cells may further enhance chlamydial replication and rescue (Whitaker and Hayw ar d 1985 ). Coincidentall y, a geneticall y modified MA104 cell line (termed MA104 N * V) was r ecentl y established for efficient isolation of rota viruses , utilizing parainfluenza virus 5 (PIV5, formerly SiV5) V protein and bovine viral diarrhea virus (BVDV) N protease to targeted degradation of signal transducer and activator of transcription 1 (ST A T1) and interfer on r egulatory factor 3 (IRF3), r espectiv el y (Rothfuc hs et al. 2006 , Peterhans andSchweizer 2013 ).Studies have shown that IRF3 is involved at an earl y sta ge in the synthesis of IFN-β induced by C. trachomatis (Hu et al. 2015 ).The JAK/ST A T signaling pathway plays a crucial role in defending against viral and intracellular bacterial infections, with human pathogens like the hepatitis C virus targeting ST A T1 and ST A T2 signaling to e v ade the immune system (Foy et al. 2003 , Rodriguez andHorvath 2004 ).In addition, upregulation of ST A T1 has been demonstrated to inhibit the growth of C. trachomatis (Lad et al. 2005 ).At present, a growing number of AIC caused by C. trac homatis genotypes D-K hav e been r eported, but fe w ocular isolates have been isolated due to limited specimens (Mohamed-Noriega et al. 2015, Petr ov ay et al. 2015 ).We propose that the culture efficiency of C. trachomatis can be greatly enhanced with the genetically modified MA104 N * V cell line.
In this study, we conducted a compar ativ e anal ysis of the culturing efficacy of HeLa, HCEC, Vero, BGMK, MA104, and MA104 N * V cells acr oss v arious bacterial concentr ations and time points.The sensitivity of MA104 N * V cells for C. trac homatis cultur e was assessed in vivo by quantifying intracytoplasmic inclusions using imm unofluor escent (IF) staining, and in vitro by quantitative PCR (qPCR).To further elucidate the potential clinical applications of MA104 N * V cell, through a comparative analysis with traditional smear cytology and qPCR, we observed that MA104 N * V cell line demonstr ated enhanced pr oficiency in cultur e identification, sub-sequent serotyping, and research applications.Expanding upon these results, we initially conducted serotype analysis on adult patients afflicted with AIC in northern China.

Cell lines
The establishment of the MA104 N * V cell line was facilitated thr ough collabor ation with the Institute of Virology, Chinese Center for Disease Control and Pr e v ention, whic h kindl y pr ovided the MA104 and MA104 N * V cell lines .T he MA104 N * V cell line was engineer ed thr ough the incor por ation of the V pr otein deriv ed fr om par a-influenza virus 5 (formerl y r eferr ed to as SiV5) and the BVDV N protease .T his strategic modification enabled the targeted inhibition of the ST A T1 and IRF3 cellular signaling pathways (Sánchez-Tacuba et al. 2020 ).In addition, we pr epar ed HeLa, HCEC (fr om ATCC), Ver o and BGMK cells (fr om the China Institute of Veterinary Drug Inspection).All cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1 × Penicillin-Stre ptom ycin solution (Gibco, USA).To stabilize the MA104 N * V cell lines, 1 μg/ml puromycin (Bey otime Biotechnology, China) w as ad ded for selecti v e r emov al of other normal MA104 cells, follo w ed b y incubation at 37 • C with 5% CO 2 .

RN A extr action and RT-qPCR
Total RN A w as extr acted fr om two cell lines, MA104 and MA104 N * V, using the RNeasy Micro Kit (QIAGEN, Hilden, Germany).RN A w as r e v erse tr anscribed into cDNA using the PrimeScript RT r ea gent Kit.Quantitativ e r eal-time PCR was conducted with LightCycler 480 SYBR Green I Master Mix (Roche, Basel, Switzerland) on a LightCycler 480 system (Roc he).Relativ e expr ession was normalized to GAPDH and calculated via the 2 − Ct method.

Western blotting
For pr oteins involv ed in the IFN expr ession pathw ay, the follo wing primary antibodies were used: IRF3 (1:1000), ST A T1 (1:1000), and  Figure 1.Sc hematic dia gr am of conjunctiv al sac swab sampling, tr ansport, stor a ge, and cultur e in clinical patients.

Chlamydia trachomatis infections
In this study, C. trachomatis Type D standard strain (NCBI number CP002054.1)was used to sie v e out the optimal method for C. trac homatis cultur e.Cells wer e seeded onto 12-well plates at a density of 1 × 10 × 5 cells per well and incubated o vernight.T he resulting confluent monolayers were inoculated with C. trachomatis Type D. Two differ ent concentr ations of bacterial solutions [1000 inclusion-forming units (IFU) and 5000 IFU] wer e pr epar ed for cell infection.The plates were incubated 1 h in 37 • C and further centrifuged at 350 × g for 0.5 h.Then, the inoculum was replaced with fresh medium supplemented with 1 mg/l cycloheximide and the studied compounds or vehicle control.

IF staining
To observe intracellular inclusion bodies after 24, 48, and 72 h, IF staining was applied .For IF staining, the cells were fixed in 4% par aformaldehyde at r oom temper atur e for 10 min.FITC-labeled monoclonal antibody (1:500; ab21211; Abcam) against the major C. trachomatis outer membrane protein was added overnight at 4 • C. Negative control was incubated without any primary antibody.The cells were followed by staining with DAPI (catalog C10005; Beyotime Biotechnology) and observed under a fluorescence micr oscope.Repr esentativ e ima ges wer e ca ptur ed with an Ol ympus micr oscope (Ol ympus BX-51, Ol ympus).

Quantifying bacterial load
Intracytoplasmic inclusions were counted using IF staining on an inv erted micr oscope using a 40x objectiv e and 20 r andoml y se-lected incubation fields after 24, 48, and 72 h of incubation.Mean v alues wer e calculated and compar ed acr oss differ ent cultur e systems .T he infected cell cultures were collected 24, 48, and 72 h after infection, and the genome copy number of C. trachomatis was determined by qPCR.

DN A extr action and qPCR
Following 24, 48, and 72 h of incubation, C. trachomatis bacterial fluid w as harvested, underw ent a freeze-thaw c ycle, and w as subsequently centrifuged at 500 × g for 10 min at 4 • C. The resulting pellet was resuspended in 2 ml of sucrose/phosphate/glutamate buffer and aliquoted before storage at −80 • C. DN A w as extracted using the DNeasy Blood & Tissue Kit (69504; QIAGEN).Quantitativ e r eal-time PCR anal yses wer e conducted using the Light-Cycler 480 SYBR Green I Master Mix on a LightCycler 480 system (Roc he).Relativ e expr ession le v els wer e normalized to GAPDH using the 2 − Ct method.HeLa cells were utilized as the reference standard for comparing the 2 − Ct ratios among various cell types in statistical analyses.Primer sequences used for amplification included: GAPDH, forw ar d 5 -GGA GCGA GA TCCCTCCAAAA T-3 , r e v erse 5 -GGCTGTTGTC AACTTCTC ATGG-3 ; and C. trachomatis , forw ar d 5 -GCCGCTTTGAGTTCTGCTTCCTC-3 , r e v erse 5 -A TTT A CGTGA GCA GCTCTCTCAT-3 .

Prospecti v e v alida tion of clinical pa tients Sample collection and culture
From February 2023 to December 2023, 20 patients suspected of AIC were examined at Beijing Tongren Hospital based on routine scr eening criteria, suc h as failur e of local tr eatment, c hr onic symptoms, clinical signs of follicular conjunctivitis, and sexual activity in young individuals .T his project was approved by the Medical Ethics Committee (TRECKY2021-024).Conjunctival swabs were collected and sent to the microbiology laboratory of Beijing Institute of Ophthalmology within 24 h for PCR, sequencing, and in vitro expansion using both conventional and optimal culture methods .T he standardized processes from sampling to detection and observation was established (Fig. 1 ).For the culture of C. trachomatis, the same six cells were used for the validation and for

Chlamydia trachomatis genotyping and phylogenetic analysis
The DN A w as extracted b y DNeasy Blood & Tissue Kit (69504, QIAGEN).The C. trachomatis w as detected b y qPCR to identify the diagnosis.Nested C. trachomatis ompA amplification was performed using the Taq DNA enzyme (MT201-02; Biomed Company, China), with primer pairs amplifying a DNA fr a gment containing the ompA gene from all C. trachomatis ompA genotypes.5 μl of DN A w as used for the first amplification with primers Yang 1 (GCCGCTTTGAG-TTCTGCTTCCTC) and Yang 2 (ATTTACGT-GA GCA GCTCTCTCAT).3 μl of product from this first round was then amplified using primers Yang 3 (TGA CTTTGTTTCGA CCGT-GTTTT) and Yang 4 (TTTTCTAGATTTC ATCTTGTTC AAT/CTG).The amplification conditions for the first round and the nested PCRs were 94 • C for 3 min, 40 cycles of 94 • C for 30 s, 56 • C for 1 min, and 72 • C for 1 min and a final extension step at 72 • C for 10 min.The final PCR products at 785 bp were analysed on a 1% a gar ose gel using GoldenVie w™ nucleic acid gel stain (EL105; Biomed Compan y).After sequencing, BLAST searc hes pr ovided by NCBI were used to compare the consensus sequences with known C. trac homatis str ain sequences.Use MEGA11 softwar e to enter Sequence alignment and phylogenetic analysis were performed.The phylogenetic tree was constructed using the ClustalW model and the maximum-likelihood method in MEGA11.The online tool ITOL was used to edit the phylogenetic tree.

Sta tistical anal ysis
The data were presented as the mean ± standard deviation from at least three independent experiments, and analysed using Gr a phP ad Prism 8.0 softwar e (Gr a phP ad, USA).Normality of quantitative data was assessed using the Shapiro-Wilk test.Differences in continuous variables between the two groups were assessed using Student's t -test or Mann-Whitney test, r espectiv el y. Between the thr ee gr oups , one-wa y ANOVA or Kruskal-Wallis test was used.Correlations between the number of intracytoplasmic inclusions and the C. trachomatis DN A cop y number w ere exam-
b Statisticall y significant differ ences in the number of intr acytoplasmic inclusions at 48 h compar ed to 24 and 72 h postinfection under consistent conditions of initial C. trachomatis concentration within the identical cells ( P < .05).
wild-type strain (both P < .05;Fig. 2 D).Taken together, these observ ations collectiv el y suggest that MA104 N * V cells exhibit reduced endogenous le v els of ST A T1 and IRF3, making them the promising cell line for isolating and culturing clinical C. trachomatis strains.

Ev alua tion of the efficiency of culturing C. trachomatis in different cell line s
Next, the C. trachomatis standard strain (genotype D) was used to infect six cell lines in vitro to e v aluate the infection efficiency of differ ent cultur e systems.In all six cell lines (Fig. 3 A), as visualized by IF staining, C. trachomatis proliferated and formed typical intracellular inclusions, with notably extensive formation in MA104 N * V cells.To determine the optimal inoculating dose, cells were infected with 1000 IFU and 5000 IFU of C. trac homatis , r espectiv el y.Notabl y, at an initial infection concentration of 5000 IFU, the number of inclusions in all six cell types was significantly higher than that in the 1000 IFU wells ( P < .001,Fig. 3 B).To r e v eal the temporal dynamics of intracytoplasmic inclusions during C. trachomatis infection, infected cells were harvested at 24, 48, and 72 h postinfection, and the number of intracytoplasmic inclusions was quantified under light microscopy.Results showed that the number of intracytoplasmic inclusions in all cell lines exhibited a trend of first increasing and then decreasing, reaching a peak at 48 h.At an initial inoculating dose of 5000 IFU, the number of intracytoplasmic inclusions in the MA104 N * V cell line peaked at 48 h, with an av er a ge of 29.9 ± 1.6 inclusions per field of view, significantly higher than the number in HeLa cells (18.2 ± 2.0 inclusions/field), HCEC cells (14.3 ± 2.0 inclusions/field), BGMK cells (19.9 ± 2.9 inclusions/field), Vero cells (19.2 ± 1.4 inclusions/field), and MA104 cells (18.1 ± 1.6 inclusions/field; Fig. 3 C).Detailed data on the dynamic changes in the number of intracytoplasmic inclusions are shown in Table 1 .Employing qPCR for mor e pr ecise quantification, the r elativ e amount of C. trachomatis was normalized to cellular GAPDH le v els.Notabl y, the MA104 N * V cell line consistently exhibited the highest C. trachomatis DNA copy number across all three assessed time points (Fig. 3 D).Additionall y, Pearson corr elation anal ysis w as performed betw een the number of intracytoplasmic inclusions and the C. trachomatis DN A cop y number.There's a strong positive correlation between the two indicators (linear r egr ession: y = 899 191 * x + 314 398; R 2 = 0.93, P < .01),indicating that the number of intracytoplasmic inclusions can be used as a surrogate indicator for C. trachomatis DN A cop y number (Fig. 3 E).Ther efor e, the number of intracytoplasmic inclusions will be used as a quantification of pathogens her eafter.Collectiv el y, these data indicate that MA104 N * V cells are the most sensitive cell line and have the highest infection efficiency.

Real-world clinical validation of the detection of C. trachomatis using MA104 N * V cell line
To further verify the clinical diagnostic value of the modified MA104 N * V cell line, which is highly susceptible to C. trachomatis , these cell lines were used to culture the superior and inferior palpebr al conjunctiv al sac specimens obtained fr om patients with suspected AIC.A total of 20 patients were included in this study.Demographic, clinical c har acteristics, and labor atory test r esults ar e detailed in Table 2 .Briefly, the median age of patients was 35 years (range 23-55 years), 60% ( n = 12) were male, and the av er a ge disease duration was 7.5 weeks.Patients showed edema and thickening of the palpebral conjuncti va, massi ve hemorrhage, and numerous follicles (80%, n = 16), particularly at the medial and lateral canthus .T he positive rate of C. trachomatis qPCR test was 35.0%(7/20).All se v en clinical samples with positiv e PCR r esults exhibited detectable intracytoplasmic inclusions in the MA104 N * V cell culture system (Fig. 4 ).In contrast, only six samples were detected in Hela and BGMK cells, and only five samples were detected in MA104 cells and Vero cells.Following the HCEC cell cultur e, onl y four samples were successfully isolated and cultured.In addition, we also e v aluated the r atio of neutr ophils and l ymphocytes in the conjunctival cell scrapings, and found that there were a relativ el y lar ge number of neutr ophils [Median (38.3%)] and a small number of lymphocytes [Median (21.5%)] infiltration.Giemsa and IF staining were performed 48 h after inoculation, and the typical cap-like structure of intracytoplasmic inclusions was found (Fig. 4 ).Notabl y, all conjunctiv al swabs that tested positive for C. trachomatis by qPCR also produced positive results in MA104 N * V cells, showing 100% concordance .T hese r esults demonstr ate that MA104 N * V has demonstrated high practicability in real-world clinical diagnosis and can provide additional laboratory evidence for the clinical diagnosis of AIC.

Phylogenetic analyses of isolated C. trachomatis strains
Finally, a total of seven C. trachomatis positive samples from patients with AIC were selected to determine their genotypes.All samples were successfully genotyped by PCR amplification, Sanger sequencing, and phylogenetic analysis of C. trachomatis ompA gene .T he genotype of all samples was first determined by BLAST and then confirmed by the phylogenetic tree constructed by after local alignment analysis (Fig. 5 ).The predominant genotype was identified as D, comprising 57.1% of the samples, n = 4), follo w ed b y E, F, and Ia, eac h r epr esenting 14.3% of the strains ( n = 1 for each).These findings contribute valuable insights into the pr e v alence and genetic div ersity of C. trac homatis str ains in   patients with AIC, with implications for understanding the epidemiological landscape of the infection.

Discussion
Globall y, C. trac homatis stands as the most pr e v alent bacterial sexuall y tr ansmitted infection, with nearl y 131 million ne w cases dia gnosed annuall y (Kasi et al. 2004, Fenwic k 2012 ).Despite its staggering impact, an estimated 4.2% of females worldwide harbor this infection, with ∼80% being asymptomatic, rendering it undiagnosed.The consequences of this undetected infection can lead to various conjunctivitis syndromes, including trachoma, adult and neonatal inclusion conjunctivitis, and l ymphogr anuloma v ener eum (Loc kington et al. 2013 ).AIC, caused by C. trac homatis genotypes D-K, r epr esents a pr e v alent ocular manifestation.Ho w e v er, the lac k of typical clinical symptoms and resemblance to other ocular diseases, such as allergic conjunctivitis, often results in underdiagnosis or misdiagnosis of AIC associated with C. trachomatis infection (Mohamed-Noriega et al. 2015 ).In this study, we successfully utilized a highly susceptible cell line with r educed IFN expr ession, MA104 N * V, to cultur e C. trac homatis and dia gnose AIC, r esulting in enhanced infection efficacy compared to commonly used cell lines.We also optimized infection conditions, including timing, concentration, and staining methods.The utilization of MA104 N * V cells r epr esents a nov el a ppr oac h to study C. trachomatis conjunctival infections, holding promise for genome-wide genetic v ariation anal ysis and e v en the de v elopment of pathogen-specific vaccines and drugs in the future.
Chlam ydia trac homatis , as a unique intr acellular pathogen, has tr aditionall y been cultur ed using McCoy, HeLa, BGMK, and Vero cell lines (Talley et al. 1994 ).Ho w e v er, the cell cultur e pr ocess is time-consuming, labor-intensiv e, and r equir es specialized facilities and expertise, posing challenges for broader implementation (Di Pietro et al. 2019 ).While PCR remains the primary clinical detection method for C. trachomatis , only cell culture allows for crucial aspects like drug sensitivity test, genetic studies, and potential v accine de v elopment (Aiyar et al. 2014 ).The primary goal of our study was to identify an impr ov ed cell line for isolation and cultur e of C. trac homatis fr om limited clinical samples to increase detection rates.In this study, our initial attempts with MA104 and MA104 N * V cells demonstrated that MA104 N * V cells, known for suppr essing IFN expr ession, yielded the highest number of intracytoplasmic inclusions at both low and high inoculum concentr ations.Notabl y, the geneticall y modified MA104 N * V cells hav e been widely used in rotavirus culture due to its ability to suppress IFN, a critical component of the host's defense a gainst v arious intracellular pathogens (Peterhans andSchweizer 2013 , Sánchez-Tacuba et al. 2020 ).Our findings corr obor ate that the degr adation of ST A T1 and IRF3, inhibiting intracellular IFN expression, enhances the infection efficacy of C. trachomatis (Rothfuchs et al. 2006 ).Recently, Carlson et al. also r e v ealed that IFN played an important role in preventing the invasion and proliferation of C. trachomatis (Carlson et al. 2004 ).Meanwhile, it has been reported that in the presence of low concentrations of indole, C. trachomatis can e v ade IFN-γ -mediated clearance, maintaining its pathogenicity (Caldwell et al. 2003, Carlson et al. 2004 ).This study introduces an innov ativ e a ppr oac h for dia gnosing AIC, successfull y isolating and cultiv ating C. trac homatis fr om clinical samples using the modified MA104 cell line.
Our study also further determined the optimal conditions for culturing C. trachomatis in vitro .We found that the peak number of intracytoplasmic inclusions occurred at 48 h postinfection, and MA104 N * V cells demonstrated the highest sensitivity to C. trachomatis among six cell lines tested.While Giemsa staining is effective for detecting intracytoplasmic inclusions, imm unofluor escence staining pr ov ed mor e pr ecise, especiall y for limited conjunctival specimens with minimal atypical intracytoplasmic inclusions (Lipkin et al. 1986 ).Our results emphasized the higher sensitivity of imm unofluor escence staining thr ough increased antigen-antibody interaction, although it comes with a higher cost and the need for additional equipment.
The successful validation of MA104 N * V cell line susceptibility using a standard D strain led us to employ this cell line for culturing C. trachomatis from clinical conjunctival sac specimens.A standar dized w orkflo w w as established covering the entir e pr ocess from sample collection to detection and observation.We also found that MA104 N * V cells are suitable for clinical detection of C. trachomatis and is consistent with the results of PCR.Moreover, we have identified four distinct genotypes of C. trachomatis , including genotype D, E, F, and Ia, consistent with the epidemiological characteristics of C. trachomatis genotypes reported globally (Stenberg and Mårdh 1990, Rao et al. 1996, Malhotra et al. 2013, Mohamed-Noriega et al. 2015 ).Additionally, our study is the first to document the genotypes with high genetic diversity of adult patients with inclusion conjunctivitis in northern China, which may prompt incr eased domestic surv eillance efforts for C. trac homatis infection and provide valuable insights for vaccine development.
Despite these contributions, our study has some limitations.First, we focus on a limited set of C. trachomatis isolates (genotype D, E, F, and Ia) due to the availability of clinical samples .T hese findings may vary with other C. trachomatis isolates, and further research is needed to confirm the superiority of the modified MA104 N * V cells.Second, the limited number of patients with conjunctivitis in our ophthalmology-focused hospital restricted the sample size and source for this study.
In conclusion, MA104 N * V cells demonstrated high sensitivity to C. trachomatis and pr ov ed effectiv e for inoculating clinical conjunctiv al samples.Imm unofluor escence staining emer ged as a valuable tool for detecting intracytoplasmic inclusions, with MA104 N * V cells exhibiting the highest intracytoplasmic inclusions count at 48 h postinfection with C. trachomatis .Notabl y, C. trac homatis genotype D was predominant in the obtained ocular conjunctival specimens.Our study provides a powerful tool for clinical isolation and culture of C. trachomatis and lays a foundation for future research on the genetic diversity of this pathogen based on whole-genome sequencing and vaccine de v elopment.
The real-time PCR protocol consisted of an initial denaturation step at 95 • C for 3 min, follo w ed b y 40 c ycles of denaturation at 95 • C for 10 s and annealing/extension at 60 • C for 30 s .T his was succeeded by a final cycle of denaturation at 95 • C for 1 min, annealing at 55 • C for 30 s, and a final denaturation at 95 • C for 30 s .T he sequences of the primers used for amplification were as follo ws: GAPDH forw ar d, 5 -GGA GCGA GA TCCCTCCAAAA T-3 and r e v erse , 5 -GGCTGTTGTC AACTTCTC A TGG-3 ; IRF3 forw ar d, 5 -A GA GGCTCGTGATGGTCAA G-3 ; and r e v erse, 5 -AGGT CC AC AGT A TTCTCCAGG-3 ; and ST A T1 forw ar d, 5 -CA GCTTGA CTCAAAATTCCTGGA-3 ; and r e v erse, 5 -TGAAGA TT ACGCTTGCTTTTCCT-3 .

F igure 2 .
Gro wth and characterization of six epithelial cell lines for culture of C. trachomatis .(A) Morphology of MA104, MA104 N * V, HCEC, Hela, BGMK, and Vero cells under contrast phase microscope.Scale bar = 100 μm.(B) Imm unofluor ence staining of ST A T1 and IRF3 within MA104 and MA104 N * V cells.Nuclei were counterstained with DAPI.Scale bar = 20 μm.(C) Western blotting demonstrating the decreased expression of ST A T1 and IRF3 in MA104 N * V cells.GAPDH was used as a loading control.(D) RT-qPCR showing the significantly decreased expression levels of ST A T1 and IRF3 in the MA104 N * V cell line ( * * P < .01,* * * P < .001). the comparison.Giemsa staining and IF staining were performed 48 h after inoculation to observe the morphology and structure of the intracytoplasmic inclusions in cell culture .T he validation of sensitive cell lines and the C. trachomatis culture process has been confirmed based on the positive rate of the clinical samples.

Figure 3 .
Figure 3. Assessments of infectivity of C. trachomatis across six epithelial cell lines.(A) Observation of six distinct cell lines 48 h post C. trachomatis infection using IF staining.Scale bar = 20 μm.(B) Comparison of the number of intracytoplasmic inclusions in six different cell lines infected with 1000 and 5000 IFU of C. trachomatis .The number of intracytoplasmic inclusions were assessed in 20 randomly selected fields at 48 h postinfection ( * * * P < .001).(C) Number of intracytoplasmic inclusions in cells infected with 5000 IFU of C. trachomatis .At 24, 48, and 72 h postinfection, 20 r andoml y selected fields were assessed for the number of intracytoplasmic inclusions in IF staining (comparison with HeLa cells, * P < .05,* * P < .01,* * * P < .001,and ns: nonsignificant).(D) C. trachomatis cultures were harvested and quantified using qPCR at 24, 48, and 72 h.Statistical comparison with HeLa cells r e v ealed significant differences ( * P < .05).(E) Scatterplot showing the strong positive correlation between the number of intracytoplasmic inclusions and the C. trachomatis DNA copy number.Pearson correlation was used.

Figure 4 .
Figure 4. Slit-lamp images from seven confirmed AIC cases, accompanied by laboratory test in MA104 N * V cells with IF and Giemsa staining.The arrow indicates typical C. trachomatis intracytoplasmic inclusions detected under the microscope.Scale bar = 50 μm.

Figure 5 .
Figure 5. Maximum-likelihood phylogenetic tree displaying the evolutionary relationships of C. trachomatis ompA gene sequences .T he lea ves of the tr ee ar e color-coded according to genotype .GenBank accession numbers of all a v ailable r efer ence sequences, indicated in par entheses, uniquel y identify each sequence in the analysis.

Table 1 .
Comparison of inclusion body counts of six kinds of cells infected with C. trachomatis at 24, 48, and 72 h under IF staining.

Table 2 .
Summary of laboratory examination results for 20 patients with suspected AIC.